During the past decade, more than 100 patients with thoracic malignancies have been treated on a series of clinical protocols examining toxicities and clinical responses following infusions of DNA demethylating agents ( Decitabine; DAC), and HDAC inhibitors, such as romidepsin (DP). Collectively these trials demonstrated no objective clinical regressions, although prolonged stabilization of disease (4-12months) was observed in approximately 10% of patients. Nearly one quarter of all patients receiving DAC infusions exhibited increased expression of p16, MAGE-3, or NY-ESO-1 in tumor tissues. Serologic responses to NY-ESO-1 were observed in several patients receiving DAC for more than six months. Approximately 50% of patients receiving DP infusions exhibited increased intratumoral levels of H3Ac and p21. In addition, several patients exhibited enhanced expression of NY-ESO-1 and MAGE-A3 in tumor biopsies following DP infusions. Micro-array analysis of laser captured tumor cells from pre and post treatment biopsies from patients receiving DAC, DP or sequential DAC/DP infusions revealed a shift from a lung cancer gene signature to one observed in normal respiratory epithelia. These findings, together with observations of up-regulation of CT-X antigens in tumor tissues provided proof of concept for the use of epigenetic regimens in combination with immunologic interventions to target CT-X antigens in thoracic malignancies. In general, thoracic malignancies exhibit low level, focal and heterogeneous CT-X gene expression, as well as variable responses to chromatin remodeling agents. Because CT-X antigens appear to be preferentially expressed in pluripotent tumor cells, it is conceivable that autologous epigenetically modified tumor cells may be unique, personalized vaccines targeting cancer stem cells, which promote systemic metastases. Recent clinical efforts have focused on the evaluation of epigenetically-modified tumor cells as adjuvant vaccines in patients with primary thoracic malignancies, as well as patients with extra-thoracic malignancies metastatic to the chest, who have been rendered NED by standard therapy. The vaccines are administered with Iscomatrix, a proprietary adjuvant that has been shown to elicit potent immunologic responses to purified CT-X antigens; additionally, oral celecoxib will be used to inhibit activity of Tregs that have been shown to attenuate immune responses to therapeutic vaccines in cancer patients. To date, 30 patients have been accrued to this trial. Despite preclinical studies demonstrating feasibility of this approach, reliable derivation of cell lines from primary tumors has been a significant challenge, in part due the histologies of the resected malignancies. Although cells lines have been established from several individuals, these patients were ineligible for vaccinations due to disease recurrence. In an attempt to circumvent the problems of establishing primary cell lines, we have identified several established lines including K562 erythroleukemia, and H1299 lung cancer cells that exhibit high level CT-X gene expression without pharmacologic manipulation for use as adjuvant vaccines. In a recent Phase II trial in the Thoracic Surgery Section, TGIB, 20 patients received six, monthly immunizations with 2.5e7-1e8 live irradiated K562 cells constitutively expressing GM-CSF (K562-GM) in conjunction with metronomic oral cyclophosphamide and celecoxib (to inhibit immunosuppressive Tregs) as adjuvant therapy following complete resection of their malignancies. Metronomic chemotherapy was well tolerated, and no vaccine-related toxicities were observed. Whereas several patients exhibited increased serologic reactivity to purified CT-X antigens following their vaccinations, none met criteria for immunologic response. Several patients with uncharacteristically long disease free intervals while on therapy, developed rapid recurrence shortly after being taken off study. These findings raise the possibility that there were responses to the vaccine that were not detected by our analysis, and/or a potential clinical benefit of the metronomic chemotherapy. A manuscript pertaining to this trial is presently being prepared for publication. In more recent efforts we have developed a vaccine using H1299 lung cancer cells. Due to amplification of the X chromosome, H1299 cells exhibit broader and higher levels of CT-X gene expression than K562-GM; as such, H1299 cells may be more effective vaccines for inducing immunity to CT antigens that potentially can be up-regulated in clinical settings by chromatin remodeling agents. However, unlike MHC-deficient K562-GM, H1299 cells exhibit class I as well as class II HLA expression; consequently, H1299 cells may induce more allo-reactivity than K562-GM cells when used as whole cell vaccines. In laboratory experiments, we have observed that relative to conventional RIPA-buffer lysates, freeze-thaw lysates of H1299 cells have significantly lower levels of HLA proteins, while retaining high levels of CT-X antigen. In a trial commencing in September 2014 (14-C-0053), thoracic oncology patients who are rendered NED by conventional therapy, yet are at high risk for recurrence, will be randomized to receive H1299 lysates with Iscomatix with or without metronomic cyclophosphamide/celecoxib. Vaccines will be administered monthly for 6 months followed by treatment evaluation one month later. A variety of biologic and immunologic endpoints will be assessed during this First-in-Humans vaccine trial.